Today, allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative treatment for many patients with hematological malignancies. Bone marrow (Aschan, 2006), peripheral mobilized stem cells (Bacigalupo et al., 2002) and umbilical cord blood (Kestendjieva et al., 2008) are the common sources for HSCT. Despite the use of highly sophisticated therapeutic approaches, HSCT is still associated with a considerable mortality caused by a number of complications such as graft versus host disease (GvHD), infectious diseases, veno-occlusive disease, donor graft rejection, and relapses of the underlying diseases.
The use of conventionally immunosuppressive drugs leads to a suppression of the entire immune system, which enhances the possibility for infections or development of malignant tumors. Also in some cases, the effectiveness of these drugs can be reduced or even abrogated. For example, steroid refractory GvHD is one of the major problems following allogeneic hematopoietic stem cell transplantation (Auletta et al., 2009; von Bonin et al., 2009). For treatment of GvHD, immunosuppressive strategies against key elements of T-cell reactions were already performed (von Bonin et al., 2009). However, because of the high numbers of patients, these strategies were mainly used in rheumatology (Kameda et al., 2009; Senolt et al., 2009) or for patients after kidney transplantation. For therapy of acute GvHD, most experiences are available for OKT3® (Benekli M et al., 2006; Knop et al., 2007) or interleukin 2 receptor antibodies (Chen et al., 2004; Ji et al., 2005), and for chronic GvHD with anti CD20 antibodies (Bates et al., 2009). However, these antibodies can be associated with less long-term success and toxicity because of appearance of infectious complications. The use of monoclonal antibodies for clinical application was restricted because of the missing humanization. Murine antibodies or antibodies from other species are huge molecules with a molecular weight in the range of 150 kDa that may be highly immunogenic in humans. After application of murine anti human monoclonal antibodies, life-threatening and anaphylactic complications were observed (Chester et al., 1995). Also, the immunogenic potential of the antibodies depends from their peptide structure. IgG4 isotypes, for example, are less immunogenic than IgG1 isotypes because of the low potential for complement activation. Besides, the humanization of antibodies leads to chimeric isotypes that are less immunogenic than their originally murine counterparts (Hosono et al., 1992). Up to date, there are no clear data that show that totally human antibodies have clinically advantages compared to chimeric antibodies.
Accordingly, the investigation of alternative or improved therapeutic approaches or procedures including the use of new cell sources, the treatment with antibodies or other biologicals are still in need.
One possible approach focuses on CD4 positive T helper cells. Said cells coordinate both the pathological and the physiological immune reaction in the human body. Influencing CD4 positive T helper cells by application of anti CD4 antibodies should, therefore, lead to a targeted modulation of the immune system.
Previously, the murine anti human CD4 monoclonal antibody Max16H5 (IgG1) was used in clinical application in patients with auto-immune diseases or as a protective therapy against transplant rejection (Chatenoud et al., 1995; Emmrich et al., 1991a; Emmrich et al., 1991b). Furthermore, in human kidney transplantation, Max16H5 (IgG1) had the potential to effectively reduce graft rejection (Reinke et al., 1991; Reinke et al., 1995). The application of anti CD4 specific monoclonal antibodies may not only result in suppression of immune activity but also in the induction of tolerance against tetanus toxoid in an triple transgeneic mouse model (Laub et al., 2002). The induction of tolerance by a rat monoclonal antibody has also been demonstrated (Kohlhaw et al., 2001). Said monoclonal antibody Max16H5 is also disclosed in EP 1 454 137, which is incorporated herein by reference and which, among others, relates to the use of a labeled ligand having specificity for the human CD4 molecule to produce an in vivo diagnostic agent.
CD4+ molecules on T helper cells bind directly to constant regions of HLA molecules on antigen presenting cells (APCs) to allow a complete T cell activation. To interfere with this binding by non depleting monoclonal antibodies may inhibit this activation by a total steric blockage, by shortening of cell-cell contact between APC and T cell (Fehervari et al., 2002) or by induction of negative signals by inhibition of protein tyrosine phosphorylation (Harding et al., 2002) or induction of T cell anergy (Madrenas et al., 1996). Here, Fehérvári at al. and Harding et al. do not disclose the methods and uses of the invention. Among others, they did not incubate stem cell grafts with anti CD4 antibodies, but isolated CD4+ cells separated out of spleens (murine) and buffy coats (human).
In addition, WO 2004/112835 describes, among others, methods involving the use of antibodies including antibodies directed against CD4. Here, anti CD4 antibodies were used to generate regulatory T cells over a long period in order to induce immunological tolerance.
In view of the above, there is still a need of promising alternative and improved, respectively, therapeutic approaches that may lack disadvantages of the prior art methodologies.
Furthermore, there are many instances whereby the efficacy of a therapeutic protein is limited by an unwanted immune reaction to the therapeutic protein. Several mouse monoclonal antibodies have shown promise as therapies in a number of human disease settings but in certain cases have failed due to the induction of significant degrees of a human anti-murine antibody (HAMA) response (Schroff et al. (1985)). For monoclonal antibodies, a number of techniques have been developed in attempt to reduce the HAMA response (see e.g. WOA9106667). These recombinant DNA approaches have generally reduced the mouse genetic information in the final antibody construct whilst increasing the human genetic information in the final construct. Notwithstanding, the resultant “humanized” antibodies have, in several cases, still elicited an immune response in patients (Isaacs J. D. (1990)).
Antibodies are not the only class of polypeptide molecule administered as a therapeutic agent against which an immune response may be mounted. Even proteins of human origin and with the same amino acid sequences as occur within humans can still induce an immune response in humans.
Key to the induction of an immune response is the presence of peptides within the protein that can stimulate the activity of T cells via presentation on MHC class II molecules, so-called “T-cell epitopes.”
MHC Class II molecules are a group of highly polymorphic proteins which play a central role in helper T cell selection and activation. The human leukocyte antigen group DR (HLA-DR) are the predominant isotype of this group of proteins; however, isotypes HLA-DQ and HLA-DP perform similar functions. In the human population, individuals bear two to four DR alleles, two DQ and two DP alleles. The structure of a number of DR molecules have been solved and these appear as an open-ended peptide binding groove with a number of pockets that engage amino acid side chains (pocket residues) of the peptide (Stern et al. (1994)). Polymorphisms identifying the different allotypes of class II molecule contributes to a wide diversity of different binding surfaces for peptides within the peptide binding groove and, at the population level, ensures maximal flexibility with regard to the ability to recognize foreign proteins and mount an immune response to pathogenic organisms.
An immune response to a therapeutic protein proceeds via the MHC class II peptide presentation pathway. Here exogenous proteins are engulfed by antigen presenting cells (APCs) and processed for presentation at the cell surface in association with MHC class II molecules of the DR, DQ or DP type. MHC Class II molecules are expressed by professional antigen presenting cells, such as macrophages and dendritic cells amongst others. Engagement of a MHC class II peptide complex by a cognate T cell receptor on the surface of the T cell, together with the cross-binding of certain other co-receptors such as the CD4 molecule, can induce an activated state within the T cell. Activation leads to the release of cytokines further activating other lymphocytes such as B cells to produce antibodies or activating T killer cells as a full cellular immune response.
T cell epitope identification is the first step to epitope elimination as recognized in WO98/52976 and WO00/34317. In these teachings, predicted T cell epitopes are removed by the use of judicious amino acid substitutions within the protein of interest. Besides computational techniques, there are in vitro methods for measuring the ability of synthetic peptides to bind MHC class II molecules. An exemplary method uses B-cell lines of defined MHC allotype as a source of MHC class II binding surface and may be applied to MHC class II ligand identification (Marshall et al. (1994); O'Sullivan et al. (1990); Robadey et al. (1997)). However, such techniques are not adapted for the screening of multiple potential epitopes to a wide diversity of MHC allotypes, nor can they confirm the ability of a binding peptide to function as a T cell epitope.
Recently, techniques exploiting soluble complexes of recombinant MHC molecules in combination with synthetic peptides have come into use (Kern et al. (1998); Kwok et al (2001)). These reagents and procedures are used to identify the presence of T cell clones from peripheral blood samples from human or experimental animal subjects that are able to bind particular MHC-peptide complexes and are not adapted for the screening multiple potential epitopes to a wide diversity of MHC allotypes.
CD4 is a surface glycoprotein primarily expressed on cells of the T lymphocyte lineage including a majority of thymocytes and a subset of peripheral T cells. Low levels of CD4 are also expressed by some non-lymphoid cells although the functional significance of such divergent cellular distribution is unknown. On mature T cells, CD4 serves a co-recognition function through interaction with MHC Class II molecules expressed in antigen presenting cells. CD4+ T cells constitute primarily the helper subset which regulates T and B cell functions during T-dependent responses to viral, bacterial, fungal and parasitic infections.
During the pathogenesis of autoimmune diseases, in particular when tolerance to self antigens breaks down, CD4+ T cells contribute to inflammatory responses which result in joint and tissue destruction. These processes are facilitated by the recruitment of inflammatory cells of the hematopoietic lineage, production of antibodies, inflammatory cytokines and mediators, and by the activation of killer cells.
CD4 antibodies are known in the art. An exemplary CD4 antibody, monoclonal mouse anti human CD4-antibody 30F16H5, is disclosed in DE 3919294. Said antibody is obtainable from the hybridoma cell line ECACC 88050502.
To reduce the immunogenicity of mouse anti-CD4 antibodies, humanized anti-CD4 antibody have been previously engineered by cloning the hypervariable regions of a mouse antibody into frameworks provided by human immunoglobulins (e.g. Boon et al. (2002)). Although reducing the immunogenicity compared to mouse anti-CD4, these humanized antibody still elicited immune responses in several cases.
Furthermore, it is known from the art that such a “humanization” of antibodies often results in antibodies with lower or significantly lower affinity to the given target.
It is, hence, a further objective of the invention to provide for modified forms of an anti human CD4-antibody to reduce the immune reaction to mouse anti-CD4 antibodies. In particular, it is desirable to provide anti-CD4 antibodies with a reduced number of T cell epitopes which may result in a reduced or absent potential to induce an immune response in a human subject. Such proteins may be expected to display an increased circulation time within a human subject capable of mounting an immune response to the non-modified antibody and may be of particular benefit in chronic or recurring disease settings such as is the case for a number of indications for anti-CD4. While others have provided anti-CD4 antibody molecules including “humanized” forms, none of these teachings recognize the importance of T cell epitopes to the immunogenic properties of the protein nor have been conceived to directly influence said properties in a specific and controlled way according to the scheme of the present invention.